Long considered strictly genetic material, DNA was shown in 1994 to be able to act as an enzyme (Breaker and Joyce 1994). Like RNAzymes, DNAzymes can catalyze nucleic acid and phosphoramidate bond cleavage, ligation, phopshorylation, and porphyrin metallation (Lu 2002). Because of their stability and catalytic capabilities, DNAzymes promise to be important in a large array of applications (Lu 2002).
Aptamers are nucleic acids (such as DNA or RNA) that recognize targets with high affinity and specificity (Ellington and Szostak 1990, Jayasena 1999). Aptazymes (also called allosteric DNA/RNAzymes or allosteric (deoxy)ribozymes) are DNA/RNAzymes regulated by an effector (the target molecule). They typically contain an aptamer domain that recognizes an effector and a catalytic domain (Hesselberth et al. 2000, Soukup and Breaker 2000, Tang and Breaker 1997). The effector can either decrease or increase the catalytic activity of the aptazyme through specific interactions between the aptamer domain and the catalytic domain. Therefore, the activity of the aptazyme can be used to monitor the presence and quantity of the effector. This strategy has been used to select and design aptazyme sensors for diagnostic and sensing purposes (Breaker 2002, Robertson and Ellington 1999, Seetharaman et al. 2001). DNA aptazymes are the most attractive candidate for sensor development because DNA is much less expensive to synthesize and more stable than RNA. In addition, general strategies to design DNA aptazymes, by introducing aptamer motifs close to the catalytic core of DNAzymes, are available (Wang et al. 2002). High cleavage activity requires the presence of effector molecules that upon binding to the aptamer motif, can allosterically modulate the activity of the catalytic core part of the aptazyme.
In vitro selection methods can be used to obtain aptamers for a wide range of target molecules with exceptionally high affinity, having dissociation constants as high as in the picomolar range (Brody and Gold 2000, Jayasena 1999, Wilson and Szostak 1999). For example, aptamers have been developed to recognize metal ions such as Zn(II) (Ciesiolka et al. 1995) and Ni(II) (Hofmann et al. 1997); nucleotides such as adenosine triphosphate (ATP) (Huizenga and Szostak 1995); and guanine(Kiga et al. 1998); co-factors such as NAD (Kiga et al. 1998) and flavin (Lauhon and Szostak 1995); antibiotics such as viomycin (Wallis et al. 1997) and streptomycin (Wallace and Schroeder 1998); proteins such as HIV reverse transcriptase (Chaloin et al. 2002) and hepatitis C virus RNA-dependent RNA polymerase (Biroccio et al. 2002); toxins such as cholera whole toxin and staphylococcal enterotoxin B (Bruno and Kiel 2002) and bacterial spores such as the anthrax (Bruno and Kiel 1999). Compared to antibodies, DNA/RNA based aptamers are easier to obtain and less expensive to produce because they are obtained in vitro in short time periods (days vs. months) and with limited cost. In addition, DNA/RNA aptamers can be denatured and renatured many times without losing their biorecognition ability. These unique properties make aptamers an idea platform for designing highly sensitive and selective biosensors (Hesselberth et al. 2000).
Radioisotope and fluorescence signals are often used to detect aptamer and aptazyme activity. Radioisotope-labeling has the advantage of minimal perturbation for the binding ability of aptamers and aptazymes (Rusconi et al. 2002, Seetharaman et al. 2001); however, safety and disposal concerns prevent this method from broad use. Fluorescence provides significant signal amplification and enables real-time monitoring of concentration fluctuations. However, determining effective parameters for using fluorophores is inefficient, requiring trial and error. If too close to the binding site, fluorophores may prevent the effector from binding; if too remote, no signal will be detected. To overcome this difficulty when using aptamers, fluorophores are incorporated into nucleotides during aptamer selection (Jhaveri et al. 2000). Many fluorophores are easily photo-bleached.
A powerful alternative to fluorophore and radio-isotope detection is colorimetry (Cao et al. 2001, Rakow and Suslick 2000, Smith et al. 1999). Colorimetric detection minimizes detection costs and safety concerns, and is well suited for on-site and real-time detection. In a colorimetric cocaine sensor based on aptamers, cocaine displaces a dye in the binding site of a cocaine aptamer (Stojanovic and Landry 2002). Because the dye has different absorption properties when bound to the aptamer, the presence of cocaine is indicated by a color change. However, finding an appropriate dye for a particular aptamer requires screening a large number of dyes. Moreover, the extinction coefficient for organic dyes seldom exceeds 106 L·mole−1·cm−1, necessitating high dye concentration for simple visual observation.
Metallic particles have extinction coefficients three orders of magnitude higher than those of organic dyes (Link et al. 1999). For effective detection, they may be used in low concentrations (nanomolar) for use as detection agents with aptamers.